Similarities between the fusion proteins of Ebola virus (EboV) and those of other RNA viruses can be used to guide strategies of drug development against this important potential bioterrorism agent. Some fusion protein motifs, including the membrane-disruptive "fusion peptide" and a pair of extended alpha-helices, have been targeted, however, combinatorial design of drugs against membrane-interactive domains of the envelope protein of EboV could be facilitated with an effective screening assay. A visual microwell plate assay, which uses the lanthanide metal terbium(III) (Tb3+) entrapped in large unilamellar phospholipid vesicles (LUV) and the aromatic chelator dipicolinic acid (DPA) for rapid, high-throughput screening for membrane-disrupting molecules, was recently developed. Preliminary results indicate that synthetic peptides corresponding to several domains of the HIV- 1 transmembrane glycoprotein (TM) interact and disrupt vesicular membranes in the Tb3+/DPA:LUV microwell assay. In Specific Aim 1 we will extend these studies to determine whether synthetic peptides corresponding to similar, potential membrane-interactive motifs of the EboV glycoprotein 2 (GP2) also mediate vesicular lysis in this assay. Synthetic peptides based on representative strains of EboV will be tested. Analog peptides will also be tested to determine if they can interfere with membrane disruption by wild-type peptides. Structural features of membrane interactive peptides will be compared to inactive analogs by various biophysical techniques, including circular dichroism spectroscopy and a novel resonance energy transfer method. Studies proposed in Specific Aim 2 will determine whether synthetic peptides corresponding to various conserved motifs of EboV GP2 can block infectivity mediated by Ebola virus glycoproteins. Pseudotyped virions with membrane-bound EboV GP and the core of murine leukemia virus will be utilized to establish the feasibility of using the Tb3+/DPA:LUV microwell assay to screen for inhibitory peptides and non-peptide inhibitors of EboV membrane interactive domains. The proposed studies will provide proof-of-concept that combinatorial chemistry approaches may be useful for developing peptide and non-peptide drugs against Ebola virus, as well as a broad range of important human viral pathogens with similar fusion proteins.